Malassezin and analogs thereof as skin brightening agents

ABSTRACT

The present invention relates to compounds, compositions, and methods for brightening skin. The compounds, compositions, and methods of the present invention generally involve compounds produced by a Malassezia yeast, and chemical analogs thereof. In addition to skin brightening applications, the compounds, compositions, and methods of the present invention may be used to modulate melanocyte activity, induce melanocyte apoptosis, agonize an arylhydrocarbon receptor (AhR), improve hyperpigmentation caused by a hyperpigmentation disorder, and modulate melanin production, melanosome biogenesis, and melanosome transfer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims benefit to U.S. provisional applicationSer. No. 62/306,468, filed Mar. 10, 2016. The entire contents of thatapplication are incorporated by reference.

FIELD OF INVENTION

The present invention relates to chemical analogs of compounds producedby a Malassezia yeast. The invention includes compositions comprisingcompounds produced by a Malassezia yeast as well as chemical analogs ofcompounds produced by a Malassezia yeast. Methods of using the compounds(including analogs thereof) and compositions of the present inventionare also contemplated.

BACKGROUND OF THE INVENTION

Individuals around the world use skin brightening agents to achieve anumber of cosmetic goals, including producing an anti-aging effect,correcting sun damage, and meeting certain cultural standards of beauty.Many commercially available skin brightening products, while effectiveto varying degrees, contain harmful ingredients, some of which have beenlinked to cancer. Thus, there exists a need for novel skin brighteningagents and formulations that exhibit higher levels of safety and/orefficacy than agents currently on the market.

Malassezia is a genus of lipophilic yeast commonly found in the normalflora of human skin. Malassezia is responsible for a number of skindiseases, including tinea versicolor (pityriasis versicolor), seborrheicdermatitis, and atopic dermatitis.

Tinea versicolor is a non-contagious skin disease caused by Malasseziaovergrowth that locally alters pigmentation levels. Malassezia yeastshave two metabolic pathways for synthesizing melanin andtryptophan-derived indole pigments. The indole pigments includemalassezin, a tryptophan metabolite of Malassezia that may elicitmelanocyte apoptosis and contribute to the depigmentation characteristicof Malassezia overgrowth.

The invention disclosed herein utilizes compounds produced by Malasseziayeast, including malassezin, and chemical analogs thereof, as the basisfor safe and efficacious skin brightening compositions.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a compound for brighteningskin. The compound is a chemical analog of a compound produced by aMalassezia yeast, or a crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound for inducingmelanocyte apoptosis. The compound is a chemical analog of a compoundproduced by a Malassezia yeast, or a crystalline form, hydrate, orcosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a compound formodulating melanocyte activity. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

An additional embodiment of the present invention is a compound foragonizing the arylhydrocarbon receptor (AhR). The compound is a chemicalanalog of a compound produced by a Malassezia yeast, or a crystallineform, hydrate, or cosmetically or pharmaceutically acceptable saltthereof.

Another embodiment of the present invention is a compound for improvinghyperpigmentation caused by a hyperpigmentation disorder. The compoundis a chemical analog of a compound produced by a Malassezia yeast, or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a compound formodulating melanin production. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

An additional embodiment of the present invention is a compound formodulating melanosome biogenesis. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound for modulatingmelanosome transfer. The compound is a chemical analog of a compoundproduced by a Malassezia yeast, or a crystalline form, hydrate, orcosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a composition. Thecomposition comprises a Malassezia yeast and a cosmetically orpharmaceutically acceptable vehicle, diluent or carrier.

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound isolated or isolatable from aMalassezia yeast and a cosmetically or pharmaceutically acceptablevehicle, diluent or carrier.

Another embodiment of the present invention is a composition. Thecomposition comprises any of the compounds, including analogs, disclosedherein and a cosmetically or pharmaceutically acceptable vehicle,diluent or carrier.

A further embodiment of the present invention is a method of brighteningskin in a subject. The method comprises contacting the subject with anyof the compounds or compositions disclosed herein.

An additional embodiment of the present invention is a method forinducing melanocyte apoptosis in a subject. The method comprisescontacting the subject with any of the compounds or compositionsdisclosed herein.

Another embodiment of the present invention is a method for modulatingmelanocyte activity in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for agonizingan arylhydrocarbon receptor (AhR) in a subject. The method comprisescontacting the subject with any of the compounds or compositionsdisclosed herein.

An additional embodiment of the present invention is a method forimproving hyperpigmentation caused by a hyperpigmentation disorder in asubject in need thereof. The method comprises contacting the subjectwith any of the compounds or compositions disclosed herein.

Another embodiment of the present invention is a method for modulatingmelanin production in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for modulatingmelanosome biogenesis in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

An additional embodiment of the present invention is a method formodulating melanosome transfer in a subject. The method comprisescontacting the subject with any of the compounds or compositionsdisclosed herein.

Another embodiment of the present invention is a compound. The compoundhas the structure of formula (II):

wherein:R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independentlyselected from the group consisting of hydrogen and methyl, and at leastone of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 is methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a compound. Thecompound has the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl, and at leastone of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

An additional embodiment of the present invention is a compound forbrightening skin. The compound has the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a compound forbrightening skin. The compound has the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a compound for inducingmelanocyte apoptosis. The compound has the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

An additional embodiment of the present invention is a compound forinducing melanocyte apoptosis. The compound has the structure of formula(III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a compound for agonizingthe arylhydrocarbon receptor (AhR). The compound has the structure offormula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a compound foragonizing the arylhydrocarbon receptor (AhR). The compound has thestructure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof, and a cosmetically or pharmaceuticallyacceptable vehicle, diluent or carrier.

Another embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof, and a cosmetically or pharmaceuticallyacceptable vehicle, diluent or carrier.

A further embodiment of the present invention is a method forbrightening skin in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

An additional embodiment of the present invention is a method forbrightening skin in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a method for agonizing anarylhydrocarbon receptor (AhR) in a subject. The method comprises:contacting the subject with a compound having the structure of formula(II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

A further embodiment of the present invention is a method for agonizingan arylhydrocarbon receptor (AhR) in a subject. The method comprises:contacting the subject with a compound having the structure of formula(III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a schematic diagram of the skin's component layers. The insetdiagram shows the cellular makeup of the epidermis and dermis. FIG. 1Bis a schematic diagram showing potential mechanisms of action ofhypopigmentation-causing agents.

FIGS. 2A-2C show synthetic schemes for malassezin and malassezinderivatives: FIG. 2A: malassezin and indolo[3,2-b] carbazole; FIG. 2B:compounds I and IV; FIG. 2C: compound II.

FIG. 3A is a summary chart showing EC₅₀ values of annexin V inductionfor certain compounds of the present invention in MeWo and WM115 cells.FIGS. 3B-3M are line graphs showing the percentage of MeWo (FIGS. 3B-3G)or VVM115 (FIGS. 3H-3M) cells labeled with annexin V after exposure tovarious concentrations of the listed compounds.

FIGS. 4A-4D are charts showing relative annexin V levels (%) in MeWo andWM115 cells after exposure to various concentrations of the listedcompounds for 6, 24, 48, and 72 hours. FIGS. 4E-4J are histogramsshowing results from FIGS. 4A-4D. FIGS. 4K and 4L are histograms showingthe percentage of MeWo (FIG. 4K) and WM115 (FIG. 4L) cells labeled withannexin V after 6-hour exposure to the listed compounds at theconcentrations shown.

FIGS. 5A-5K are micrographs showing MeWo cell morphology after 6 hoursof treatment with various concentrations of CV-8684, CV-8685, CV-8688,DMSO, and staurosporine.

FIGS. 6A-6K are micrographs showing MeWo cell morphology after 24 hoursof treatment with various concentrations of CV-8684, CV-8685, CV-8688,DMSO, and staurosporine.

FIGS. 7A-7K are micrographs showing MeWo cell morphology after 48 hoursof treatment with various concentrations of CV-8684, CV-8685, CV-8688,DMSO, and staurosporine.

FIGS. 8A-8K are micrographs showing MeWo cell morphology after 72 hoursof treatment with various concentrations of CV-8684, CV-8685, CV-8688,DMSO, and staurosporine.

FIGS. 9A-9K are micrographs showing WM115 cell morphology after 6 hoursof treatment with various concentrations of CV-8684, CV-8685, CV-8688,DMSO, and staurosporine.

FIGS. 10A-10K are micrographs showing WM115 cell morphology after 24hours of treatment with various concentrations of CV-8684, CV-8685,CV-8688, DMSO, and staurosporine.

FIGS. 11A-11K are micrographs showing WM115 cell morphology after 48hours of treatment with various concentrations of CV-8684, CV-8685,CV-8688, DMSO, and staurosporine.

FIGS. 12A-12K are micrographs showing WM115 cell morphology after 72hours of treatment with various concentrations of CV-8684, CV-8685,CV-8688, DMSO, and staurosporine.

FIGS. 13A-13D are charts showing the percentage of viable MeWo and WM115cells remaining after treatment with various concentrations of CV-8684(FIG. 13A), CV-8685 (FIG. 13B), CV-8688 (FIG. 13C), or staurosporine(FIG. 13D) for 6, 24, 48, and 72 hours. Cell viability was assayed usingCellTiter-Glo®.

FIGS. 13E-13J are histograms showing results from FIGS. 13A-13D. FIG.13K is a summary chart comparing percentages of viable MeWo and WM115cells after exposure to the listed concentrations of malassezin,indolocarbazole, compound II, and staurosporine for 24, 48, and 72hours.

FIGS. 14A-14D are charts showing levels of lactate dehydrogenase (“LDH”)release from MeWo and WM115 cells after treatment with variousconcentrations of CV-8684 (FIG. 14A), CV-8685 (FIG. 14B), CV-8688 (FIG.14C), or staurosporine (FIG. 14D) for 6, 24, 48, and 72 hours. FIGS.14E-14J are histograms showing results from FIGS. 14A-14D. FIGS. 14K and14L are histograms showing lactate dehydrogenase levels after exposingMeWo (FIG. 14K) and WM115 (FIG. 14L) cells to the listed concentrationsof malassezin, carbazole, compound II, and staurosporine for 24 hours.

FIGS. 15A-15E show raw data and line graphs of arylhydrocarbon receptor(“AhR”) activation in HepG2 cells stably transfected with anAhR-responsive luciferase reporter gene plasmid upon exposure to variousconcentrations of omeprazole (FIG. 15A), CV-8684 (FIG. 15B), CV-8685(FIG. 15C), CV-8686 (FIG. 15D), and CV-8688 (FIG. 15E). FIG. 15F showsEC₅₀ values for each compound tested.

FIGS. 16A-16K are photographs of MelanoDerm™ matrices at either day 0 orday 7 after exposure to no treatment (FIG. 16A), sterile deionized water(FIG. 16B), 1% kojic acid (FIG. 16C), 0.2% DMSO (FIG. 16D), 0.05% DMSO(FIG. 16E), 200 μM CV-8684 (FIG. 16F), 50 μM CV-8684 (FIG. 16G), 200 μMCV-8686 (FIG. 16H), 50 μM CV-8686 (FIG. 16I), 200 μM CV-8688 (FIG. 16J),and 50 μM CV-8688 (FIG. 16K).

FIGS. 17A-17K are 15× magnification photomicrographs of MelanoDerm™matrices at either day 0 or day 7 after exposure to no treatment (FIG.17A), sterile deionized water (FIG. 17B), 1% kojic acid (FIG. 17C), 0.2%DMSO (FIG. 17D), 0.05% DMSO (FIG. 17E), 200 μM CV-8684 (FIG. 17F), 50 μMCV-8684 (FIG. 17G), 200 μM CV-8686 (FIG. 17H), 50 μM CV-8686 (FIG. 17I),200 μM CV-8688 (FIG. 17J), and 50 μM CV-8688 (FIG. 17K).

FIGS. 18A-18F are photographs of zebrafish exposed to no treatment (FIG.18A), DMSO (FIG. 18B), phenylthiourea (“PTU”) (FIG. 18C), and compoundII at 2.5 μM (FIG. 18D), 5 μM (FIG. 18E), and 10 μM (FIG. 18F). Redarrows indicate normal melanocytes.

FIGS. 19A-19F are photographs of zebrafish exposed to no treatment (FIG.19A), DMSO (FIG. 19B), phenylthiourea (“PTU”) (FIG. 19C), and compoundII at 0.3 μM (FIG. 19D), 1 μM (FIG. 19E), and 3 μM (FIG. 19F). Redarrows indicate normal melanocytes. Yellow arrows indicate abnormallysmall melanocytes.

FIG. 20 is a summary chart showing the number and percent of zebrafishwith decreased skin pigmentation after exposure to the listedconditions. The final six rows show the effects of variousconcentrations of compound II.

FIGS. 21A-21E are photographs of zebrafish treated with no treatment(FIG. 21A), DMSO (FIG. 21B), PTU (FIG. 21C), 0.5 μM (FIG. 21D), and 1.5μM (FIG. 21E). Bottom panels include regions of color scheme inversion.

FIGS. 22A and 22B are histograms showing pigmentation density asmeasured by pigmented pixels/mm³ (FIG. 22A) and total pixels (FIG. 22B)from photographs of zebrafish embryos, exemplified in FIGS. 21A-21E.

FIGS. 23A-23C are mass spectra of CV-8684 in DMSO (FIG. 23A), RPMI media(FIG. 23B), and DMEM (FIG. 23C). FIGS. 23D-23F are mass spectra ofCV-8686 in DMSO (FIG. 23D), RPMI media (FIG. 23E), and DMEM (FIG. 23F).FIGS. 23G-23I are mass spectra of CV-8688 in DMSO (FIG. 23G), RPMI media(FIG. 23H), and DMEM (FIG. 23I). FIG. 23J is a summary chart showingpercent of test compound remaining in the listed solvent after 2-hourincubation.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a compound for brighteningskin. The compound is a chemical analog of a compound produced by aMalassezia yeast, or a crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof.

As used herein, the term “compound” refers to two or more atoms that areconnected by one or more chemical bonds. In the present invention,chemical bonds include, but are not limited to, covalent bonds, ionicbonds, hydrogen bonds, and van der Waals interactions. Covalent bonds ofthe present invention include single, double, and triple bonds.Compounds of the present invention include, but are not limited to,organic molecules.

Organic compounds/molecules of the present invention include linear,branched, and cyclic hydrocarbons with or without functional groups. Theterm “C_(x-y)” when used in conjunction with a chemical moiety, such as,alkyl, alkenyl, alkynyl or alkoxy is meant to include groups thatcontain from x to y carbons in the chain. For example, the term “C_(x-y)alkyl” means substituted or unsubstituted saturated hydrocarbon groups,including straight-chain alkyl and branched-chain alkyl groups thatcontain from x to y carbons in the chain, including haloalkyl groupssuch as trifluoromethyl and 2,2,2-trifluoroethyl, etc. The terms“C_(x-y) alkenyl” and “C_(x-y) alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but containing atleast one double or triple bond respectively.

The term “aliphatic”, as used herein, means a group composed of carbonand hydrogen atoms that does not contain aromatic rings. Accordingly,aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclylgroups.

The term “alkyl” means the radical of saturated aliphatic groups thatdoes not have a ring structure, including straight chain alkyl groups,and branched chain alkyl groups.

The term “alkenyl”, as used herein, means an aliphatic group containingat least one double bond.

The term “alkynyl”, as used herein, means an aliphatic group containingat least one triple bond.

As used herein, an “aromatic compound”, “aromatic”, or compoundcontaining an “aromatic ring” is an aryl or a heteroaryl compound. Theterm “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 3- to 8-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike. The term “heteroaryl” includes substituted or unsubstitutedaromatic single ring structures, preferably 3- to 8-membered rings, morepreferably 5- to 7-membered rings, even more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, indole,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine,and pyrimidine, and the like. Preferably, certain compounds of thepresent invention include at least one, preferably two, indole groups aswell as at least one aldehyde group.

The term “substituted” means moieties having at least one substituentthat replaces a hydrogen atom on one or more carbons of the backbone. Itwill be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance with thepermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. The permissible substituents can be oneor more and the same or different for appropriate organic compounds.

As used herein, “skin brightening” and grammatical variations thereofrefers generally to any actual or perceived reduction in skinpigmentation. Skin brightening methods have been used to reducepigmentation of hyperpigmented areas of skin resulting from age, sunexposure, or a hyperpigmentation disorder. Application of the compoundsand compositions of the present invention to, for example, a subject'sskin, can reduce pigmentation so that the skin appears lighter or whiterthan before said application. Skin pigmentation can be assessed in anumber of ways, including, but not limited to, visual assessments using,for example, the von Luschan chromatic scale, the Fitzpatrick skintyping test (Fitzpatrick et al., 1988) and the Taylor HyperpigmentationScale (Taylor et al., 2005) and reflectance spectrophotometry methods(Zonios, et al., 2001). For example, the Fitzpatrick skin typing testincludes six types of skin (I-VI), and Type VI skin that becomes Type Vor less has been “brightened” as the term is used herein. As discussedfurther below, skin brightening can result due to a number of phenomena,including, but not limited to, modulation of melanocyte activity,induction of melanocyte apoptosis, agonism of an arylhydrocarbonreceptor (AhR), or modulation of melanin production, melanosomebiogenesis, or melanosome transfer.

Certain compounds of the present invention are produced by, isolatedfrom, or isolatable from a Malassezia yeast. Malassezia yeasts areyeasts of the genus Malassezia and include, but are not limited to,Malassezia globosa, Malassezia restricta, Malassezia furfur, Malasseziasympodialis, Malassezia slooffiae, Malassezia obtusa, Malasseziapachydermatis, Malassezia dermatis, Malassezia japonica, Malassezianana, Malassezia yamatoensis, Malassezia equine, Malassezia caprae, andMalassezia cuniculi. (Guého, et al., 1996; Gaitanis, et al., 2013).Malassezia yeast are part of the normal human cutaneous flora andtypically produce no pathogenic effects. However, Malassezia yeast cancause a number of diseases, including, but not limited to pityriasisversicolor (both the hyperpigmented and hypopigmented varieties),seborrheic dermatitis, dandruff, atopic dermatitis, Malasseziafolliculitis, psoriasis, and confluent and reticulated papillomatosis.(Gaitanis, et al., 2013).

As used herein, the term “chemical analog” refers to a compound that isstructurally related to a parent compound and contains differentfunctional groups or substituents. For example, a parent compound of thepresent invention is malassezin, and chemical analogs of malassezincontain certain functional groups and substituents that are distinctfrom malassezin. Chemical analogs of the present invention may havesignificant advantages over a given parent compound, including apharmacokinetic profile suitable for cosmetic use. In some embodiments,a chemical analog is generated from a parent molecule by one or morechemical reactions. In other embodiments, alternative synthesis schemesthat do not originate with a parent compound can be used to generatechemical analogs of the present invention.

A compound of the present invention is “produced by a Malassezia yeast”if, over the course of its lifecycle, a Malassezia yeast wouldsynthesize, secrete, accumulate, or otherwise generate the compoundunder appropriate growth conditions. Malassezia yeast secrete differentcompounds depending on what their growth media is supplemented with.(Nazzaro-Porro, et al., 1978). The present invention includes anycompound produced by a Malassezia yeast under any growth condition, butpreferred compounds include, for example, malassezin and chemicalanalogs thereof.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

Malassezin is one example of a compound produced by a Malassezia yeastof the present invention. Malassezin, also known as2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde, is a tryptophanmetabolite originally isolated from Malassezia furfur. Malassezin is aknown agonist of the arylhydrocarbon receptor (AhR), a receptorimplicated in cell growth, differentiation, and gene expression. (Willeet al., 2001). Malassezin also induces apoptosis in primary humanmelanocytes. (Krämer, et al., 2005). Recently, certain chemical analogsof malassezin were synthesized by Winston-McPherson and colleagues, whoexamined the analogs' AhR agonist activity. (Winston-McPherson, et al.,2014).

Another embodiment of the present invention is a compound for inducingmelanocyte apoptosis. The compound is a chemical analog of a compoundproduced by a Malassezia yeast, or a crystalline form, hydrate, orcosmetically or pharmaceutically acceptable salt thereof.

As used herein, the term “melanocyte” refers to a dendritic cell of theepidermis that normally synthesizes tyrosinase and, within melanosomes,the pigment melanin. Melanocytes of the present invention exhibitupregulation of certain genes, including, but not limited to, one ormore of the following: tyrosinase (oculocutaneous albinism IA),microphthalmia-associated transcription factor, alpha-2-macroglobulin,tyrosinase-related protein 1, solute carrier family 16, GS3955 protein,v-kit Hardy-Zuckerman 4 feline sarcoma, ocular albinism 1, Rag Dprotein, glycogenin 2, G-protein-coupled receptor, family C,oculocutaneous albinism II, deleted in esophageal cancer 1, melan-A,SRY-box 10, ATPase, Class V, type 10C, matrix metalloproteinase 1,latent transforming growth factor beta b, ATP-binding cassette,sub-family C, hydroxyprostaglandin dehydrogenase 15, transmembrane 7superfamily member 1, glutaminyl-peptide cyclotransferase, and othergenes identified by Lee and colleagues. (Lee, et al., 2013).

Melanocytes, like many other cell types, undergo programmed cell deathor, apoptosis. Melanocyte apoptosis pathways are known to those of skillin the art (Wang, et al., 2014), and apoptosis pathways generally havebeen reviewed by Elmore (Elmore, 2007). A compound or composition of thepresent invention “induces” melanocyte apoptosis by, for example,causing the activation of certain pro-apoptotic signal transductionpathways or causing the repression of certain anti-apoptotic pathways ina melanocyte. It is envisioned that the compound or composition of thepresent invention can directly activate/repress an apoptosis-relatedpathway by directly interacting with a signaling molecule of the pathwayor by indirectly interacting with a molecule of the pathway via directinteraction with one or more intermediary molecules that do nottypically function within the pathway.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

A further embodiment of the present invention is a compound formodulating melanocyte activity. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

Melanocyte activity can be modulated in a number of ways contemplated inthe present invention, including, but not limited to, inducingmelanocyte apoptosis or altering melanocyte gene expression, cellmotility, cell growth, melanin production, melanosome biogenesis, ormelanosome transfer.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

An additional embodiment of the present invention is a compound foragonizing the arylhydrocarbon receptor (AhR). The compound is a chemicalanalog of a compound produced by a Malassezia yeast, or a crystallineform, hydrate, or cosmetically or pharmaceutically acceptable saltthereof.

As used herein, the terms “agonist”, “agonizing”, and grammaticalvariations thereof refer to a molecule that triggers (e.g., initiates orpromotes), partially or fully enhances, stimulates or activates one ormore biological activities. Agonists of the present invention includenaturally occurring substances as well as synthetic substances.

An arylhydrocarbon receptor (AhR) of the present invention is anyarylhydrocarbon receptor that naturally exists in a subject as describedherein. Arylhydrocarbon receptors are known to those of skill in theart. (Noakes, 2015). Agonists of arylhydrocarbon receptors include, butare not limited to, tryptophan-related compounds such as kynurenine,kynurenic acid, cinnabarinic acid, and 6-formylindolo [3,2-b] carbazole(FICZ). Malassezin is also known as an aryl hydrocarbon receptoragonist. (Wille, et al., 2001).

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

Another embodiment of the present invention is a compound for improvinghyperpigmentation caused by a hyperpigmentation disorder. The compoundis a chemical analog of a compound produced by a Malassezia yeast, or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

As used herein, the compounds, compositions, and methods of the presentinvention can be used to improve hyperpigmentation caused by ahyperpigmentation disorder by, for example, reducing the level ofhyperpigmentation in areas affected by a hyperpigmentation disorder,slowing further hyperpigmentation, or preventing furtherhyperpigmentation from occurring. However, because every subject may notrespond to a particular dosing protocol, regimen, or process, improvinghyperpigmentation caused by a hyperpigmentation disorder does notrequire that the desired physiologic response or outcome be achieved ineach and every subject or subject population. Accordingly, a givensubject or subject population may fail to respond or respondinadequately to dosing, but other subjects or subject populations mayrespond and, therefore, experience improvement in theirhyperpigmentation disorder.

As used herein, the term “hyperpigmentation” is an actual or a perceivedskin disorder of excessive dark color. The skin impairment can beactual, for example, attributed to age, excessive sun exposure, or adisease or condition leading to dark skin areas. The dark skin areas canbe in the form of spots, blotches, or relatively large areas of darkcolor. The skin impairment also can be perceived, for example, aperception by an individual that his/her skin shade is too dark. Theindividual may have a cosmetic desire to lighten the skin shade.

Hyperpigmentation disorders are disorders in which hyperpigmentation isthe primary symptom as well as disorders in which hyperpigmentationoccurs as a secondary symptom. Hyperpigmentation disorders of thepresent invention include, but are not limited to, congenitalhyperpigmentation disorders and acquired hyperpigmentation disorders.Congenital hyperpigmentation disorders of the present invention include,but are not limited to, those involving epidermal hyperpigmentation(nevus cell nevus, Spitz nevus, and nevus spilus), dermalhyperpigmentation (blue nevus, nevus Ohta, dermal melanosis, nevus Ito,and Mongolian spot), ephelides, acropigmentation reticularis,Spitzenpigment/acropigmentation, and lentiginosis (generalizedlentiginosis, LEOPARD syndrome, inherited patterned lentiginosis, Carneycomplex, Peutz-Jeghers syndrome, Laugier-Hunziker-Baran syndrome, andCronkhite-Canada syndrome). (Yamaguchi, et al., 2014). Acquiredhyperpigmentation disorders of the present invention include, but arenot limited to, senile lentigines/lentigo, melasma/chloasma, Riehl'smelanosis, labial melanotic macule, penile/vulvovaginal melanosis,erythromelanosis follicularis faciei Kitamura, UV-induced pigmentation(tanning and pigmentation petaloides actinica), postinflammatorypigmentation (friction melanosis and ashy dermatosis),chemical/drug-induced pigmentation (polychlorinated biphenyl, arsenic,5-FU, bleomycin, cyclophosphamide, methotrexate, chlorpromazine,phenytoin, tetracycline, and chloroquine), pigmentary demarcation lines,and foreign material deposition (such as carotene, silver, gold,mercury, bismuth, and tattoos). Hyperpigmentation related with systemicdisorders includes metabolism/enzyme disorders (hemochromatosis,Wilson's disease, Gaucher's disease, Niemann-Pick's disease,amyloidosis, ochronosis, acanthosis nigricans, and porphyria cutaneatarda), endocrine disorders (Addison's disease, Cushing syndrome, andhyperthyroidism), nutritional disorders (pellagra, vitamin B12deficiency, folic acid deficiency, vagabond's disease, and prurigopigmentosa), mastocytosis, collagen diseases, liver dysfunction, andkidney dysfunction. Hyperpigmentation can also be related withinfectious diseases (measles, syphilis, and Malassezia furfur) andsyndromes (von Recklinghausen's disease, Sotos syndrome, POEMS syndrome,Naegeli syndrome, Cantu syndrome, McCune-Albright syndrome, Watsonsyndrome, and Bloom syndrome). (Yamaguchi, et al., 2014).

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

A further embodiment of the present invention is a compound formodulating melanin production. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

Melanin is a naturally produced pigment that gives color to skin andhair. A schematic diagram of the skin is shown in FIG. 1A. Melanin isproduced by melanocytes in organelles known as melanosomes. A compoundor composition of the present invention modulates melanin production ina subject by, for example, modulating melanosome biogenesis and directlyor indirectly inhibiting melanin synthesis at the enzymatic level.

Melanosome biogenesis occurs via four stages: Stage I is characterizedby pre-melanosomes, which are essentially non-pigmented vacuoles. Instage II, pre-melanosomes develop striations on which melanin isdeposited in stage III. Stage IV results in mature melanosomes that arerich in melanin content. Compounds and compositions of the presentinvention modulate melanosome biogenesis by inhibiting or attenuatingthe biological processes that normally promote any or all of thesestages. (Wasmeier, et al., 2008).

Melanin synthesis primarily involves three enzymes: tyrosinase,tyrosinase related protein-1, and dopachrome tautomerase. Additionalfactors that affect intracellular trafficking of these enzymes include,but are not limited to, BLOC-1, OA1, and SLC45A2. The compounds andcompositions of the present invention can modulate melanin productionby, for example, inhibiting or attenuating the activity of any of theseenzymes or factors. (Yamaguchi, et al., 2014).

Once melanosomes have formed and melanin has been synthesized,melanosomes need to be transferred from epidermal melanocytes to skinand hair keratinocytes. Melanosomes originate near the nucleus ofmelanocytes and are transported to the periphery of melanocytes alongmicrotubules and actin filaments. Compounds and compositions of thepresent invention modulate melanosome transfer by interfering with anyof the biological processes that result in the transport of melanosomesfrom the perinuclear region, to the melanocyte periphery, and intoadjacent keratinocytes. A schematic diagram of melanin synthesis,melanin transport, and melanocyte apoptosis is shown in FIG. 1B.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

An additional embodiment of the present invention is a compound formodulating melanosome biogenesis. The compound is a chemical analog of acompound produced by a Malassezia yeast, or a crystalline form, hydrate,or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

Another embodiment of the present invention is a compound for modulatingmelanosome transfer. The compound is a chemical analog of a compoundproduced by a Malassezia yeast, or a crystalline form, hydrate, orcosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound produced by a Malasseziayeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogof malassezin.

A further embodiment of the present invention is a composition. Thecomposition comprises a Malassezia yeast and a cosmetically orpharmaceutically acceptable vehicle, diluent or carrier.

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound isolated or isolatable from aMalassezia yeast and a cosmetically or pharmaceutically acceptablevehicle, diluent or carrier.

A compound isolated from a Malassezia yeast of the present inventionnecessarily exists, before isolation, in a Malassezia yeast or isproduced by a Malassezia yeast. Therefore, a compound isolated from aMalassezia yeast is derived from actual yeast cells. Standard protocolsfor extracting compounds from cellular material are known to those ofskill in the art.

A compound isolatable from a Malassezia yeast need not be derived fromactual yeast cells. Instead, synthetic reactions can be used to generatecompounds produced in yeast without the involvement of actual yeastcells. Organic synthesis reactions are well known to those of skill inthe art and can be used in this regard.

Another embodiment of the present invention is a composition. Thecomposition comprises any of the compounds disclosed herein, includinganalogs, and a cosmetically or pharmaceutically acceptable vehicle,diluent or carrier.

A further embodiment of the present invention is a method of brighteningskin in a subject. The method comprises contacting the subject with anyof the compounds or compositions disclosed herein.

As used herein, the term “contacting” and grammatical variations thereofrefer to bringing two or more materials into close enough proximity thatthey can interact. Thus, for illustrative purposes only, a compound ofthe present invention can contact a melanocyte by, for example,interacting with a receptor on the surface of the melanocyte. Similarly,a composition of the present invention can contact a human subject by,for example, being applied directly to the subject's skin.

As used herein, a “subject” means a mammalian cell, tissue, organism, orpopulations thereof. Subjects of the present invention are preferablyhuman, including human cells, tissues, and beings, but otherwiseinclude, primates, farm animals, domestic animals, laboratory animals,etc. Some examples of agricultural animals include cows, pigs, horses,goats, etc. Some examples of domestic animals include dogs, cats, etc.Some examples of laboratory animals include primates, rats, mice,rabbits, guinea pigs, etc.

An additional embodiment of the present invention is a method forinducing melanocyte apoptosis in a subject. The method comprisescontacting the subject with any of the compounds or compositionsdisclosed herein.

Another embodiment of the present invention is a method for modulatingmelanocyte activity in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for agonizingan arylhydrocarbon receptor (AhR). The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

An additional embodiment of the present invention is a method forimproving hyperpigmentation caused by a hyperpigmentation disorder in asubject in need thereof. The method comprises contacting the subjectwith any of the compounds or compositions disclosed herein.

As used herein, a subject “in need” of improvement in hyperpigmentationcaused by a hyperpigmentation disorder includes subjects with a real orperceived need of improvement.

Another embodiment of the present invention is a method for modulatingmelanin production in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for modulatingmelanosome biogenesis in a subject. The method comprises contacting thesubject with any of the compounds or compositions disclosed herein.

An additional embodiment of the present invention is a method formodulating melanosome transfer in a subject. The method comprisescontacting the subject with any of the compounds or compositionsdisclosed herein.

Another embodiment of the present invention is a compound. The compoundhas the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl,and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 ismethyl; or a crystalline form, hydrate, or cosmetically orpharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

A further embodiment of the present invention is a compound. Thecompound has a structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl, and at leastone of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is:

An additional embodiment of the present invention is a compound forbrightening skin. The compound has the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

Another embodiment of the present invention is a compound forbrightening skin. The compound has the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

A further embodiment of the present invention is a compound for inducingmelanocyte apoptosis. The compound has the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

An additional embodiment of the present invention is a compound forinducing melanocyte apoptosis. The compound has the structure of formula(III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

Another embodiment of the present invention is a compound for agonizingthe arylhydrocarbon receptor (AhR). The compound has the structure offormula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

A further embodiment of the present invention is a compound foragonizing the arylhydrocarbon receptor (AhR). The compound has thestructure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

An additional embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of formula (II)

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof, and a cosmetically or pharmaceuticallyacceptable vehicle, diluent or carrier.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

Another embodiment of the present invention is a composition. Thecomposition comprises a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof, and a cosmetically or pharmaceuticallyacceptable vehicle, diluent or carrier.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

A further embodiment of the present invention is a method forbrightening skin in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

An additional embodiment of the present invention is a method forbrightening skin in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

Another embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

A further embodiment of the present invention is a method for inducingmelanocyte apoptosis in a subject. The method comprises: contacting thesubject with a compound having the structure of formula (III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

An additional embodiment of the present invention is a method foragonizing an arylhydrocarbon receptor (AhR) in a subject. The methodcomprises: contacting the subject with a compound having the structureof formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a crystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

Another embodiment of the present invention is a method for agonizing anarylhydrocarbon receptor (AhR) in a subject. The method comprises:contacting the subject with a compound having the structure of formula(III):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independentlyselected from the group consisting of hydrogen and methyl; or acrystalline form, hydrate, or cosmetically or pharmaceuticallyacceptable salt thereof.

In one aspect of this embodiment, the compound is selected from thegroup consisting of:

As used herein, the term “composition” means an entity comprising acompound of the present invention, as well as any entity which results,directly or indirectly, from combinations of a compound of the presentinvention with other ingredients. Compositions of the present inventioncan be used as, for example, in vitro or in vivo research reagents.Compositions of the present invention can also be applied directly tothe skin of a human or non-human subject for a cosmetic effect.

A composition of the present invention may be administered in anydesired and effective manner: for oral ingestion or for parenteral orother administration in any appropriate manner such as intraperitoneal,subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal,vaginal, sublingual, intramuscular, intravenous, intraarterial,intrathecal, or intralymphatic. Further, a composition of the presentinvention may be administered in conjunction with other compositions. Acomposition of the present invention may be encapsulated or otherwiseprotected against gastric or other secretions, if desired.

The compositions of the invention comprise one or more activeingredients in admixture with one or more cosmetically orpharmaceutically acceptable carriers and, optionally, one or more othercompounds, ingredients and/or materials. Regardless of the route ofadministration selected, the compounds and compositions of the presentinvention are formulated into cosmetically or pharmaceuticallyacceptable dosage forms by conventional methods known to those of skillin the art.

Cosmetically or pharmaceutically acceptable vehicles, diluents andcarriers are well known in the art and include materials suitable forcontact with the tissues of humans and non-humans without unduetoxicity, incompatibility, instability, irritation, allergic responseand the like. Cosmetically or pharmaceutically acceptable vehicles,diluents and carriers include any substantially non-toxic substanceconventionally usable, for example, for topical, oral, peritoneal, orsubcutaneous administration of cosmetics or pharmaceuticals in which thecompounds and compositions of the present invention will remain stableand bioavailable when applied, injested, injected, or otherwiseadministered to a human or non-human subject. Cosmetically orpharmaceutically acceptable carriers suitable for topical applicationare known to those of skill in the art and include cosmetically orpharmaceutically acceptable liquids, creams, oils, lotions, ointments,gels, or solids, such as conventional cosmetic night creams, foundationcreams, suntan lotions, sunscreens, hand lotions, make-up and make-upbases, masks and the like. Carriers suitable for a selected dosage formand intended route of administration are well known in the art, andacceptable carriers for a chosen dosage form and method ofadministration can be determined using ordinary skill in the art.

The compositions of the present invention can contain other ingredientsconventional in cosmetics including perfumes, estrogen, Vitamins A, Cand E, alpha-hydroxy or alpha-keto acids such as pyruvic, lactic orglycolic acids, lanolin, vaseline, aloe vera, methyl or propyl paraben,pigments and the like. Non-limiting cosmetically or pharmaceuticallyacceptable vehicles, diluents and carriers of the present inventioninclude sugars (e.g., lactose, sucrose, mannitol, and sorbitol),starches, cellulose preparations, calcium phosphates (e.g., dicalciumphosphate, tricalcium phosphate and calcium hydrogen phosphate), sodiumcitrate, water, aqueous solutions (e.g., saline, sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection), alcohols (e.g., ethylalcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol,propylene glycol, and polyethylene glycol), organic esters (e.g., ethyloleate and triglycerides), biodegradable polymers (e.g.,polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)),elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ,olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes(e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc.

The compositions of the invention may, optionally, contain additionalingredients and/or materials commonly used in cosmetic compositions.These ingredients and materials are well known in the art and include,for example, (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monostearate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Compositions of the present invention suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, powders,granules, a solution or a suspension in an aqueous or non-aqueousliquid, an oil-in-water or water-in-oil liquid emulsion, an elixir orsyrup, a pastille, a bolus, an electuary or a paste. These formulationsmay be prepared by methods known in the art, e.g., by means ofconventional pan-coating, mixing, granulation or lyophilizationprocesses.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or more cosmetically orpharmaceutically acceptable carriers and, optionally, one or morefillers, extenders, binders, humectants, disintegrating agents, solutionretarding agents, absorption accelerators, wetting agents, absorbents,lubricants, and/or coloring agents. Solid compositions of a similar typemay be employed as fillers in soft and hard-filled gelatin capsulesusing a suitable excipient. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared using a suitable binder, lubricant, inertdiluent, preservative, disintegrant, surface-active or dispersing agent.Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in the cosmeticformulating art. They may also be formulated so as to provide slow orcontrolled release of the active ingredient therein. They may besterilized by, for example, filtration through a bacteria-retainingfilter. These compositions may also optionally contain opacifying agentsand may be of a composition such that they release the active ingredientonly, or preferentially, in a certain portion of the gastrointestinaltract, optionally, in a delayed manner. The active ingredient can alsobe in microencapsulated form.

Liquid dosage forms for oral administration include cosmetically orpharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Compositions of the present invention for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing one or more active ingredient(s) with one or more suitablenonirritating carriers which are solid at room temperature, but liquidat body temperature and, therefore, will melt in the rectum or vaginalcavity and release the active compound. Compositions of the presentinvention which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such cosmetically or pharmaceutically acceptable carriers asare known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops, emulsions, suspensions, aerosols, and inhalants. Anydesired conventional vehicles, assistants and optionally further activeingredients may be added to the formulation.

Preferred assistants originate from the group comprising preservatives,antioxidants, stabilisers, solubilisers, vitamins, colorants, odourimprovers, film formers, thickeners and humectants.

Solutions and emulsions can comprise the conventional vehicles, such assolvents, solubilisers and emulsifiers, for example water, ethanol,isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butyl glycol, oils, in particularcottonseed oil, groundnut oil, maize oil, olive oil, castor oil andsesame oil, glycerol fatty acid esters, polyethylene glycols and fattyacid esters of sorbitan, or mixtures of these substances.

The emulsions may exist in various forms. Thus, they can be, forexample, an emulsion or microemulsion of the water-in-oil (W/O) type orof the oil-in-water (O/W) type, or a multiple emulsion, for example ofthe water-in-oil-in-water (W/O/W) type.

The compositions according to the invention may also be in the form ofemulsifier-free, disperse preparations. They can be, for example,hydrodispersions or Pickering emulsions.

Suspensions may comprise conventional vehicles, such as liquid diluents,for example water, ethanol or propylene glycol, suspension media, forexample ethoxylated isostearyl alcohols, polyoxyethylene sorbitol estersand polyoxyethylene sorbitan esters, microcrystalline cellulose,aluminium metahydroxide, bentonite, agar-agar and tragacanth, ormixtures of these substances.

Pastes, ointments, gels and creams may comprise conventional vehicles,for example animal and vegetable fats, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures of thesesubstances.

Face and body oils may comprise the conventional vehicles, such assynthetic oils, such as fatty acid esters, fatty alcohols, siliconeoils, natural oils, such as vegetable oils and oily plant extracts,paraffin oils, lanolin oils, or mixtures of these substances.

Sprays may comprise the conventional propellants, for examplechlorofluorocarbons, propane/butane or dimethyl ether.

Compositions of the present invention suitable for parenteraladministrations comprise one or more compounds in combination with oneor more cosmetically or pharmaceutically acceptable sterile isotonicaqueous or non-aqueous solutions, dispersions, suspensions or emulsions,or sterile powders which may be reconstituted into sterile injectablesolutions or dispersions just prior to use, which may contain suitableantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient, or suspending or thickeningagents. Proper fluidity can be maintained, for example, by the use ofcoating materials, by the maintenance of the required particle size inthe case of dispersions, and by the use of surfactants. Thesecompositions may also contain suitable adjuvants, such as wettingagents, emulsifying agents and dispersing agents. It may also bedesirable to include isotonic agents. In addition, prolonged absorptionof the injectable cosmetic form may be brought about by the inclusion ofagents which delay absorption.

In some cases, in order to prolong the effect, it is desirable to slowits absorption from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility.

The rate of absorption of the active agent/drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered composition may be accomplished by dissolvingor suspending the active composition in an oil vehicle. Injectable depotforms may be made by forming microencapsule matrices of the activeingredient in biodegradable polymers. Depending on the ratio of theactive ingredient to polymer, and the nature of the particular polymeremployed, the rate of active ingredient release can be controlled. Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue. Theinjectable materials can be sterilized for example, by filtrationthrough a bacterial-retaining filter.

The compositions of the present invention may be presented in unit-doseor multi-dose sealed containers, for example, ampules and vials, and maybe stored in a lyophilized condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the typedescribed above.

In the present invention, the term “crystalline form” means the crystalstructure of a compound. A compound may exist in one or more crystallineforms, which may have different structural, physical, pharmacological,or chemical characteristics. Different crystalline forms may be obtainedusing variations in nucleation, growth kinetics, agglomeration, andbreakage. Nucleation results when the phase-transition energy barrier isovercome, thereby allowing a particle to form from a supersaturatedsolution. Crystal growth is the enlargement of crystal particles causedby deposition of the chemical compound on an existing surface of thecrystal. The relative rate of nucleation and growth determine the sizedistribution of the crystals that are formed. The thermodynamic drivingforce for both nucleation and growth is supersaturation, which isdefined as the deviation from thermodynamic equilibrium. Agglomerationis the formation of larger particles through two or more particles(e.g., crystals) sticking together and forming a larger crystallinestructure.

The term “hydrate”, as used herein, means a solid or a semi-solid formof a chemical compound containing water in a molecular complex. Thewater is generally in a stoichiometric amount with respect to thechemical compound.

As used herein, “cosmetically or pharmaceutically acceptable salt”refers to a derivative of the compounds disclosed herein wherein thecompounds are modified by making acid or base salts thereof. Examples ofcosmetically or pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. For example, such salts include salts from ammonia,L-arginine, betaine, benethamine, benzathine, calcium hydroxide,choline, deanol, diethanolamine (2,2′-iminobis(ethanol)), diethylamine,2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine,N-ethyl-glucamine, hydrabamine, 1H-imidazole, lysine, magnesiumhydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassiumhydroxide, 1-(2-hydroxy-ethyl)-pyrrolidine, sodium hydroxide,triethanolamine (2,2′,2″-nitrilotris(ethanol)), trometh-amine, zinchydroxide, acetic acid, 2.2-dichloro-acetic acid, adipic acid, alginicacid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoicacid, 2,5-dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoricacid, (+)-camphor-10-sulfonic acid, carbonic acid, cinnamic acid, citricacid, cyclamic acid, decanoic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid,2-hydroxy-ethanesulfonic acid, ethylenediamonotetraacetic acid, formicacid, fumaric acid, galacaric acid, gentisic acid, D-glucoheptonic acid,D-gluconic acid, D-glucuronic acid, glutamic acid, glutantic acid,glutaric acid, 2-oxo-glutaric acid, glycero-phosphoric acid, glycine,glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid,hydrochloric acid isobutyric acid, DL-lactic acid, lactobionic acid,lauric acid, lysine, maleic acid, (−)-L-malic acid, malonic acid,DL-mandelic acid, methanesulfonic acid, galactaric acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, octanoic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid(embonic acid), phosphoric acid, propionic acid, (−)-L-pyroglutamicacid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearicacid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid,thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. Furthercosmetically or pharmaceutically acceptable salts can be formed withcations from metals like aluminum, calcium, lithium, magnesium,potassium, sodium, zinc and the like.

The cosmetically or pharmaceutically acceptable salts of the presentinvention can be synthesized from a compound disclosed herein whichcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a sufficient amount of the appropriatebase or acid in water or in an organic diluent like ether, ethylacetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.

It is envisioned that the compounds and compositions of the presentinvention may be included in cosmetic or pharmaceutical compositions.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods

Isolation of Compounds Produced by Malassezia

Malassezin is isolated using, for example, the procedures outlined inWille et al., 2001. The protocol is briefly outlined below.

Medium

A medium consisting of Tween 80 (30 mL), cycloheximide (0.5 g),chloramphenicol (0.05 g), agar (20 g), and a volume of water sufficientfor a 1000 mL mixture is sterilized and mixed with 0.3% sterile filteredL-tryptophan at a concentration of 0.3 g % at 50° C. 10 mL portions arepoured into 10 cm Petri dishes and the pH is adjusted to 5.5 using 0.1 MHCl.

Cultivating Malassezia furfur and Isolating Compounds Produced By M.furfur

Malassezia furfur is swabbed on the medium described above and incubatedfor 14 days at 30° C. The contents of the Petri dish are pureed andextracted with ethyl acetate for 12 hours. The extract is filtered overglass wool, evaporated to dryness, and dissolved in methanol. Theextract is then fractionated by chromatography on Sephadex LH-20 withmethanol as the eluent. Further separation is accomplished withpreparative thin-layer chromatography with toluene:ethyl formate:formicacid (10:5:3). Main zones are partitioned between water and ethylacetate. Fractions are analyzed for activity of interest. Compounds fromfractions of interest are isolated by HPLC.

Synthesis of Malassezin and Chemical Analogs of Malassezin

Malassezin is synthesized according to the protocol set forth in Willeet al., 2001. Chemical analogs of malassezin are synthesized accordingto novel synthesis protocols, as well as those described inWinston-McPherson, et al., 2014.

Screening Protocols

Effective skin brightening compounds are evaluated using both screeningprotocols known to those of skill in the art and novel screeningmethods. For example, malassezin and chemical analogs thereof areevaluated by a tyrosinase bioassay, as described above. Other screeningprotocols involving both in vitro cell and in vivo tissue models areutilized, including aryl hydrocarbon receptor (AhR) binding assays.

Tyrosinase Bioassay

Tyrosinase bioassays are performed as described in Wille et al., 2001.Briefly, L-DOPA is mixed with tyrosinase enzyme. Extinction is measuredover 1 minute, indicating the formation of dopaquinone. Using, forexample, the fractions discussed above, these fractions are dissolved inDMSO and added directly to the tyrosinase reaction, with pure DMSO as acontrol. Tyrosinase inhibitory activity is measured as reduced increasein extinction compared to control.

Aryl Hydrocarbon Receptor Binding Assay

AhR binding assays are performed according to the protocol described in,for example, Song, et al., 2002. Briefly, human and murine AhRs areexpressed in vitro using, for example, a TnT Quick-coupled ReticulocyteLysate Systems reaction (Promega, Madison, Wis.). Receptor ligandbinding studies utilize velocity sedimentation on sucrose gradients asdescribed in Karchner, et al., 1999.

EROD Assay

Compounds, compositions, and formulations of the present invention arealso evaluated using the ethoxyresorufin-O-deethylase (EROD) assay knownto those of skill in the art. (Donato, et al., 1993; Whyte, et al.,2000; Wille et al., 2001).

Melanocyte Apoptosis Assays

Candidate compounds are evaluated for apoptosis-inducing activity inmelanocytes. Human epidermal melanocytes are cultured in Medium 254supplemented with Human Melanocyte Growth Supplement (HMGS)(Thermo-Fisher Scientific, Waltham, Mass.) or Dermal Cell Basal Medium(ATCC, Manassas, Va.). Additional components of human melanocyte growthmedia can include, but are not limited to, insulin (5 μg/ml), ascorbicacid (50 μg/ml), L-glutamine (6 mM), epinephrine (1.0 μM), and calciumchloride (0.2 mM). Human melanocyte cultures are maintained at 37° C. in5% CO₂.

Candidate compounds are diluted in DMSO and mixed directly intomelanocyte cultures. Equivalent volumes of pure DMSO are used ascontrols. Cytotoxicity assays known to those of skill in the art areperformed according to manufacturer's instructions. Cytotoxicity assaysthat are used in the present invention include, but are not limited to,CellTox™ Green Cytotoxicity Assay, Apo-ONE fluorescent caspase assays,ApoTox-Glo™ assay, and Caspase-Glo® assays (Promega, Madison, Wis.).Fluorescence detection is accomplished using standard FACS or microscopyassays known to those in the art, including those described in Krämer,et al., 2005.

Additional means of assessing apoptosis are used, including FACSanalyses for annexin V and Western blots for caspase-9 expression.Western blotting is performed according to methods known to those ofskill in the art.

Mouse Xenograft Assays

Mouse xenograft models of human skin are generated according toprotocols known in the art. (Black, et al., 1985; Manning et al., 1973;Reed, et al., 1973; Plenat, et al., 1992; Scott et al., 1998;Otulakowski, et al., 1994). Once established, mouse xenograft models areexposed to compounds of the present invention and changes inpigmentation are observed as compared to controls. Changes in skinpigmentation are assessed using various pigmentation scales known tothose of skill in the art, including, but not limited to, theFitzpatrick skin typing test and the Taylor Hyperpigmentation Scale.(Taylor, et al., 2005).

Human Assays

Compounds, compositions, and formulations of the present invention areapplied to humans, for example, on human skin, and compared to controlsubstances. Changes in skin pigmentation are assessed using variouspigmentation scales known to those of skill in the art, including, butnot limited to, the Fitzpatrick skin typing test and the TaylorHyperpigmentation Scale.

Example 2 Biochemical Target of Malassezin and Its Analogs

It is expected that the compounds and compositions of the presentinvention will exhibit, for example, tyrosinase inhibition and AhRagonist activity comparable to malassezin. Compounds and compositions ofthe present invention are expected to exhibit, for example, more potenttyrosinase inhibition and stronger AhR agonism compared to malassezin.Likewise, certain of the compounds and compositions of the presentinvention are expected to be less effective tyrosinase inhibitors andAhR agonists than malassezin. Such compounds, compositions, andformulations may have more favorable toxicity profiles compared to morepotent compounds.

Example 3 In Vitro Efficacy

It is expected that the compounds and compositions of the presentinvention will induce melanocyte apoptosis and modulate melanocyteactivity, melanin production, melanosome biogenesis, and/or melanosometransfer at least as potently as malassezin. It is also contemplatedthat certain of the compounds and compositions of the present inventionwill effect these biological processes less potently than malassezin.Such compounds and compositions may have more favorable toxicityprofiles compared to more potent species.

Example 4 In Vivo Efficacy

It is expected that the compounds and compositions of the presentinvention will be at least as effective as malassezin for brighteningskin and improving hyperpigmentation caused by hyperpigmentationdisorders. It is further expected that the compounds and compositions ofthe present invention will exhibit favorable pharmacokinetic profiles interms of, for example, half-life and absorption. Certain compounds willexhibit a longer half-life, whereas others will exhibit a shorterhalf-life. Similarly, certain compounds will exhibit differentabsorption profiles, with some compounds taking longer to be fullyabsorbed and others taking less time to be fully absorbed.

Example 5 Synthesis of Malassezin and Malassezin Derivatives

Malassezin (“CV-8684”) and its cyclized derivative indolo[3,2-b]carbazole (“CV-8685”) were synthesized according to the scheme shown inFIG. 2A.

Synthesis of tert-butyl (2-iodo-phenyl)carbamate, Compound 1

To a solution of 2-iodo-aniline (25.0 g, 0.114 mol) in tetrahydrofuran(250 mL) at 0° C. was added LiHMDS (251.0 mL, 1 M in THF, 0.251 mol)slowly while maintaining the internal temperature below 5° C. over 40min. After 30 min stirring at 0° C., a solution of BOC anhydride (27.0g, 0.125 mol) in THF (50 mL) was slowly added while maintaining theinternal temperature below 5° C. over 40 min. The reaction mixture waswarmed to ambient temperature and stirred 1 hr. Saturated NH₄Cl (250 mL)was added to quench the reaction. The organic layer was separated andwashed with water (150 mL). The combined aqueous layer was extractedwith ethyl acetate (2×150 mL), the layers were separated. The ethylacetate layer was combined with the original organic layer andconcentrated in vacuo to give as brown oil. The crude compound purifiedby column chromatography (0-5% ethyl acetate/hexanes). Compound 1 wasobtained as a light yellow liquid (29.0 g, 80%).

Synthesis of Compound 2

Copper iodide (0.95 g, 10% mol) and PdCl₂(PPh₃)₄ (1.75 g, 5% mol) wasadded to a degassed solution of compound 1 (16.0 g, 0.05 mol), propargylmethyl ether (4.25 g, 0.06 mol) in triethylamine (200 mL) at ambienttemperature. After stirring at ambient temperature over 2 hr, thereaction was complete (monitored by TLC using 10% ethylacetate/hexanes). The reaction mixture diluted with ethyl acetate (300mL), reaction mixture was washed with water, saturated NaCl and driedover Na₂SO₄. The solvent was filtered and concentrated in vacuo to giveas brown oil. The crude compound purified by column chromatography (10%ethyl acetate/hexane). Compound 2 was obtained as a light yellow liquid(13.0 g, 99%).

Synthesis of Compound 3

To an oven-dried flask was added PtCl2 (0.26 g, 0.001 mol), Na2CO3 (1.6g, 0.015 mol), indole (2.32 g, 0.02 mol) and compound 2 (2.6 g, 0.01mol) in dioxane (120 mL). The flask was degassed with nitrogen, sealedand heated to 100° C. overnight. After the reaction was complete(monitored by TLC using 10% ethyl acetate/hexanes). The solvent wasevaporated under reduced pressure. The reaction mixture diluted withethyl acetate (200 mL), reaction mixture was washed with water,saturated NaCl and dried over Na₂SO₄. The solvent was filtered andconcentrated in vacuo to give as brown oil.

This reaction was repeated using compound 2 (2.6 g, 0.01 mol) indifferent batch. Both batches crude compounds were combined and purifiedby column chromatography (10% ethyl acetate/hexane). Compound 3 wasobtained as a light brown solid (3.8 g, 55%).

Synthesis of Compound 4

Potassium carbonate (4.6 g, 0.0329 mol) was added to a solution ofcompound 3 (3.8 g, 0.0109 mol) in methanol (150 mL) and water (50 mL)mixture at ambient temperature. The resulting suspension was heated toreflux overnight. After the reaction was complete (monitored by TLCusing 20% ethyl acetate/hexanes). The reaction mixture was cooled toambient temperature and solvent concentrated in vacuo. The residue takenin ethylacetate (200 mL) and washed with water and brine then dried(sodium sulfate), filtered, solvent concentrated in vacuo to give as abrown solid. Crude compound purified by column chromatography (20% ethylacetate/hexane. Compound 4 was obtained as an orange color solid (2.2 g,81%).

Synthesis of Compound Malassezin (CV-8684)

To a dried 100 rnL two neck round-bottom flask under argon at 0° C.dimethylformamide (20 mL) was added. POCl₃ (0.75 g, 0.0048 mol) slowlyadded while maintaining the internal temperature below 5° C. over 10min. After 30 min stirring at 0° C., a solution of compound 4 (1.0 g,0.004 mol) in dimethylformamide (5 mL) was slowly added whilemaintaining the internal temperature below 5° C. over 10 min. Theresulting mixture was stirred at ambient temperature overnight. Afterthe reaction was complete (monitored by TLC using 20% ethylacetate/hexanes). The reaction mixture was poured into saturated aqueoussodium bicarbonate (150 mL) and stirred for 1 hr. Resulting mixture wasextracted with ethyl acetate (2×100 mL). The organic layers werecombined and washed with water, saturated NaCl and dried over Na₂SO₄.The solvent was filtered and concentrated in vacuo to give as brownsolid. The crude compound purified by column chromatography (0-20% ethylacetate/hexanes). Compound Malassezin (CV-8684) was obtained as a lightpink solid (0.82 g, 74%).

HPLC purity: 97.8% (area %). ¹H-NMR, ¹³C spectrum consistent with thestructure. ESI-MS: Calc. for C₁₈H₁₅N₂O (M+H)⁺: 275, found: 275.2.

Synthesis of Compound Indolo[3,2-b] Carbazole (CV-8685).

Concentrated HCl (0.25 mL) was added to a solution of malassezin (0.75g) in tetrahydrofuran (120 mL) at ambient temperature. The resultingmixture was heated to reflux overnight. After the reaction was complete(monitored by TLC using 40% ethyl acetate/hexanes). The reaction mixturewas cooled to ambient temperature and stirred for 1 hr. Filtered thesolid, washed with tetrahydrofuran (20 mL) and dried to giveIndolo[3,2-b] carbazole (CV-8685) light yellow solid (0.55 g, 78%).

HPLC purity: 96.22% (area %). ¹H-NMR, ¹³C spectrum consistent with thestructure. ESI-MS: Calc. for C₁₈H₁₃N₂ (M+H)⁺: 257, found: 257.5.

Compound I (“CV-8686”) and compound IV (“CV-8687”) were synthesizedaccording to the scheme shown in FIG. 2B.

Synthesis of Compound 5

To an oven-dried flask was added PtCl2 (1.0 g, 0.0038 mol), Na2CO3 (6.1g, 0.057 mol), 6-methyl indole (10.0 g, 0.076 mol) and compound 2 (10.0g, 0.038 mol) in dioxane (250 mL). The flask was degassed with nitrogen,sealed and heated to 100° C. overnight. After the reaction was complete(monitored by TLC using 10% ethyl acetate/hexanes). The solvent wasevaporated under reduced pressure. The reaction mixture diluted withethyl acetate (400 mL), reaction mixture was washed with water,saturated NaCl and dried over Na₂SO₄. The solvent was filtered andconcentrated in vacuo to give as brown oil. Crude compound purified bycolumn chromatography (10% ethyl acetate/hexane). Compound 5 wasobtained as a light brown solid (6.5 g, 47%).

Synthesis of Compound 6

Potassium carbonate (7.4 g, 0.054 mol) was added to a solution ofcompound 5 (6.5 g, 0.018 mol) in methanol (150 mL) and water (50 mL)mixture at ambient temperature. The resulting suspension was heated toreflux overnight. After the reaction was complete (monitored by TLCusing 20% ethyl acetate/hexanes). The reaction mixture was cooled toambient temperature and solvent concentrated in vacuo. The residue takenin ethylacetate (200 mL) and washed with water and brine then dried(sodium sulfate), filtered, solvent concentrated in vacuo to give asbrown solid. Crude compound purified by column chromatography (20% ethylacetate/hexane). Compound 6 was obtained as an orange color solid (3.3g, 72%).

Synthesis of Compound Compound I (CV-8686)

To a dried 100 mL two neck round-bottom flask under argon at 0° C.dimethylformamide (20 mL) was added. POCl₃ (0.6 g, 0.0038 mol) slowlyadded while maintaining the internal temperature below 5° C. over 10min. After 30 min stirring at 0° C., a solution of compound 6 (1.0 g,0.0038 mol) in dimethylformamide (5 mL) was slowly added whilemaintaining the internal temperature below 5° C. over 10 min. Theresulting mixture was stirred at ambient temperature overnight. Afterthe reaction was complete (monitored by TLC using 20% ethylacetate/hexanes). The reaction mixture was poured into saturated aqueoussodium bicarbonate (150 mL) and stirred for 1 hr. Resulting mixture wasextracted with ethyl acetate (2×100 mL). The organic layers werecombined and washed with water, saturated NaCl and dried over Na₂SO₄.The solvent was filtered and concentrated in vacuo to give as brownsolid. The crude compound purified by column chromatography (0-20% ethylacetate/hexanes). Compound I (CV-8686) was obtained as a light pinksolid (0.84 g, 75%).

HPLC purity: 97.01% (area %). ¹H-NMR, ¹³C spectrum consistent with thestructure. ESI-MS: Calc. for C₁₉H₁₇N₂O (M+H)⁺: 289, found: 289.1.

Synthesis of Compound Compound IV (CV-8687)

Concentrated HCl (0.3 mL) was added to a solution of compound I (1.0 g)in tetrahydrofuran (125 mL) at ambient temperature. The resultingmixture was heated to reflux overnight. After the reaction was complete(monitored by TLC using 40% ethyl acetate/hexanes). The reaction mixturewas cooled to ambient temperature and stirred for 1 hr. Filtered thesolid, washed with tetrahydrofuran (20 mL) and dried to give compound IV(CV-8687) light yellow solid (0.84 g, 89%).

HPLC purity: 98.4% (area %). ¹H-NMR, ¹³C spectrum consistent with thestructure. ESI-MS: Calc. for C₁₉H₁₅N₂ (M+H)⁺: 271, found: 271.3.

Compound II (“CV-8688”) was synthesized according to the scheme shown inFIG. 2C.

Synthesis of Compound 7

Copper iodide (0.53 g, 10% mol) and PdCl₂(PPh₃)₄ (1.0 g, 5% mol) wasadded to a degassed solution of compound 1 (9.0 g, 0.03 mol),3-methoxy-1-butyne (2.8 g, 0.035 mol) in triethylamine (150 mL) atambient temperature. After stirring at ambient temperature over 2 hr.The reaction was complete (monitored by TLC using 10% ethylacetate/hexanes). The reaction mixture diluted with ethyl acetate (300mL), reaction mixture was washed with water, saturated NaCl and driedover Na₂SO₄. The solvent was filtered and concentrated in vacuo to giveas brown oil. The crude compound purified by column chromatography (10%ethyl acetate/hexane). Compound 7 was obtained as a light yellow liquid(7.0 g, 90%).

Synthesis of Compound 8

To an oven-dried flask was added PtCl2 (0.68 g, 0.0025 mol), Na2CO3 (4.0g, 0.038 mol), indole (6.0 g, 0.05 mol) and compound 7 (10.0 g, 0.025mol) in dioxane (250 mL). The flask was degassed with nitrogen, sealedand heated to 100° C. overnight. After the reaction was complete(monitored by TLC using 10% ethyl acetate/hexanes). The solvent wasevaporated under reduced pressure. The reaction mixture diluted withethyl acetate (400 mL), reaction mixture was washed with water,saturated NaCl and dried over Na₂SO₄. The solvent was filtered andconcentrated in vacuo to give as brown oil. Crude compound purified bycolumn chromatography (10% ethyl acetate/hexane). Compound 8 wasobtained as a light brown solid (3.5 g, 77%).

Synthesis of Compound 9

Potassium carbonate (3.8 g, 0.027 mol) was added to a solution ofcompound 8 (3.3 g, 0.0091 mol) in methanol (75 mL) and water (25 mL)mixture at ambient temperature. The resulting suspension was heated toreflux overnight. After the reaction was complete (monitored by TLCusing 20% ethyl acetate/hexanes). The reaction mixture was cooled toambient temperature and solvent concentrated in vacuo. The residue takenin ethylacetate (200 mL) and washed with water and brine then dried(sodium sulfate), filtered, solvent concentrated in vacuo to give asbrown solid. Crude compound purified by column chromatography (20% ethylacetate/hexane). Compound 9 was obtained as an orange color solid (2.1g, 88%).

Synthesis of Compound Compound II (CV-8688)

To a dried 100 mL two neck round-bottom flask under argon at 0dimethylformamide (20 was added. POCl₃ (0.76 g, 0.005 mol) slowly addedwhile maintaining the internal temperature below 5° C. over 10 min.After 30 min stirring at 0° C., a solution of compound 9 (1.3 g, 0.005mol) in dimethylformamide (5 mL) was slowly added while maintaining theinternal temperature below 5° C. over 10 min. The resulting mixture wasstirred at ambient temperature overnight. After the reaction wascomplete (monitored by TLC using 20% ethyl acetate/hexanes). Thereaction mixture was poured into saturated aqueous sodium bicarbonate(150 mL) and stirred for 1 hr. Resulting mixture was extracted withethyl acetate (2×100 mL). The organic layers were combined and washedwith water, saturated NaCl and dried over Na₂SO₄. The solvent wasfiltered and concentrated in vacuo to give as brown solid. The crudecompound crystallized in chloroform (25 mL). Compound II (CV-8688) wasobtained as a light pink solid (0.81 g, 53%).

HPLC purity: 98.94% (area %). ¹H-NMR, ¹³C spectrum consistent with thestructure. ESI-MS: Calc. for C₁₉H₁₇N₂O (M+H)⁺: 289, found: 289.0.

Example 6 Cell Morphology

Typical cell morphology after various treatments is shown in FIGS.5A-5K, 6A-6K, 7A-7K, 8A-8K, 9A-9K, 10A-10K, 11A-11K, and 12A-12K. Themorphology of both cell lines was significantly affected by 100 μM ofCV-8684 and CV-8688, as well as staurosporine treatment at 6 hours.CV-8685 appeared to only affect WM115 at 100 μM.

Example 7 Apoptosis-Inducing Activity of Malassezin and MalassezinDerivatives—Preliminary Annexin V Assays

Materials and Reagents

Annexin V-FITC assay kit was purchased from Beyotime Biotechnology, RPMI1640 medium and Dulbecco's modified Eagle medium (“DMEM”) were purchasedfrom Gibco, fetal bovine serum (“FBS”) was purchased from Invitrogen,stabilized antibiotic antimycotic solution (100×) was purchased fromSigma, and 0.25% trypsin-EDTA (1×), phenol red was purchased fromInvitrogen.

Cell Culture

MeWo (ATCC® HTB-65™) and WM115 (ATCC® CRL-1675) cells were purchasedfrom ATCC (Manassas, Va.) and maintained in the following: for MeWo:DMEM supplemented with 10% FBS; for WM115: RPMI 1640 supplemented with10% FBS (10% FBS, 1% stabilized antibiotic anti-mycotic solution).

Study Summary

In the intermediate stages of apoptosis, phosphatidylserine (“PS”) istranslocated from the inner to the outer leaflet of the cell membrane,exposing PS to the extracellular environment, where it can be detected.Highly fluorescent annexin V conjugates provide quick and reliabledetection methods for studying the externalization of PS.

During the first set of studies, both MeWo and WM115 cells were treatedwith test compounds at 10 doses starting from 100 μM with 3-folddilution. Staurosporine was used as positive control. After 6-hourtreatment, cell apoptosis was assessed using an annexin V assay. Thetest compounds evaluated were CV-8684, CV-8685, CV-8686, CV-8687, andCV-8688.

Assay Procedures

For cell seeding, cells were harvested and the cell number wasdetermined using Countess® cell counter. Cells were then diluted withculture medium to the desired density. 40 μL of cell suspension per wellwas added to the required number of wells in a 384-well plate (Corning3712—clear bottom plate). The final cell density was 6,000 cells/well.After plating, the plates were incubated at 37° C. and 5% CO₂ overnight.

For preparation of compound source plate, each test compound wasdissolved in DMSO to 10 mM stock. 3-fold serial dilution was performedusing an EVO200™ liquid handler (TECAN) to generate ten concentrationsof test compound. 0.1% DMSO was employed as vehicle (negative) control.The compound source plate was then spun at room temperature at 1,000 RPMfor 1 minute and agitated using a plate shaker for 2 minutes.

For compound treatment, 40 nL of compound were transferred from thecompound source plate to the 384-well culture plate using liquid handlerEcho550 (LabCyte Inc.). After 6-hour incubation, the plates were removedfrom the incubator for detection.

For the preliminary annexin V assay, the plates were removed from theincubator and allowed to equilibrate at room temperature for 15 minutes.Culture media was then removed. 20 μL of pre-mixed annexin V-FITC andHoechst33342 dye working solution were added to each well. The cellswere then incubated at room temperature for 20 minutes. The plates weresealed and centrifuged for 1 minute at 1,000 RPM to remove bubbles.Afterward, the plate was read using an Acumen eX3 plate reader. Therelative activity was calculated according to the following formula:Activity (%)=100%×(Count_(Annexin V)/Count_(Total cell)), and EC₅₀ wascalculated using GraphPad Prism (v. 5.01).

Results

In the preliminary screen discussed above, CV-8688 markedly increasedannexin V staining of MeWo cells, with an EC₅₀ of 908.57 nM.Staurosporine, the positive control, greatly increased annexin Vstaining in both cell lines. (FIGS. 3A-3M).

Example 8 Apoptosis-Inducing Activity of Malassezin and MalassezinDerivatives—Additional Evaluation Using Annexin V Assays

Study Summary

To further investigate the impact of test compounds on apoptosis,multiple readouts, covering different stages of apoptosis, were carriedout on both MeWo and WM115 cells. Both cell types were treated with testcompounds at 3 doses (100 μM. 10 μM, and 1 μM). Staurosporine was usedas a positive control. After the desired treatment period (6, 24, 48, or72 hours), apoptosis was assessed by measuring percentages of cellsdemonstrating annexin V binding after exposure to the test compounds.The test compounds evaluated were CV-8684, CV-8685, and CV-8688.

Assay Procedures

Cell seeding was performed as discussed above with the followingexceptions: the final cell density was 4,000 cells/well for 6-hour and24-hour detections, whereas 2,000 cells/well were used for 48-hour and72-hour detections. For each time point, 384-well clear bottom plates(Corning 3712) and solid white bottom plates (Corning 3570) wereprepared. The plates were incubated as discussed above.

For preparation of the compound source plate, each test compound wasdissolved in DMSO to 10 mM stock. Two additional concentrations weregenerations by 10-fold dilution to 1 mM and 0.1 mM. Staurosporine wasused as positive control and 1% DMSO was employed as vehicle (negative)control. The compound source plate was spun at room temperature at 1,000RPM for 1 minute and agitated using a plate shaker for 2 minutes.

400 nL of test compound was transferred from the compound source plateto 384-well culture plates using Echo550 liquid handler. After 6, 24,48, and 72 hours, the plates were removed from the incubator fordetections.

For the annexin V assay, plates were removed from the incubator andequilibrated at room temperature for 15 minutes. Culture media wasremoved and cells were washed twice with PBS. 20 μL of pre-mixed annexinV-FITC working solution was added to each well. The cells were incubatedat room temperature for 20 minutes. Plates were read using Acumen eX3 tocount the number of FITC-positive cells. The relative activity wascalculated according to the following formula: Relative Activity(%)=100%×(Count_(sample)/Count_(vehicle)).

Results

CV-8684 induced apoptosis at the highest concentration tested after 6hours of treatment on both MeWo and WM115 cells. CV-8685 showed theinduction effect with 24 hours of treatment on WM115, whereas 48 hoursof treatment appeared to elicit apoptosis in both cell types. Finally,CV-8688 showed the induction effect within 6 hours of treatment in adose-dependent manner in both cell types. (FIGS. 4A-4L).

Example 9 Cell Viability After Exposure to Malassezin and MalassezinDerivatives—CellTiter-Glo® Assays

Assay Procedures

CellTiter-Glo® 2.0 assay was purchased from Promega. Cell seeding,preparation of the compound source plate, and exposure of cells to testcompounds were performed as described in Example 8.

For the CellTiter-Glo® assay, plates were removed from the incubator andequilibrated at room temperature for 15 minutes. CellTiter-Glo® reagentswere thawed and equilibrated to room temperature before the experiment.40 μL of CellTiter-Glo® reagent was then added to each well fordetection (at 1:1 ratio to culture medium). The plates were thenincubated at room temperature for 30 minutes and read using EnSpire(PerkinElmer) plate reader. The remaining activity was calculatedaccording to the following formula:Remaining Activity(%)=100%×(Lum_(sample)−Lum_(bkg))/(Lum_(vehicle)−Lum_(bkg)).Results

CV-8684 showed dose-dependent inhibition of cell viability in both celllines tested, though the inhibitory effect appeared to be more potent inMeWo cells. CV-8685 exhibited the inhibitory effect on WM115 cellviability in a dose-dependent manner only after 24-hour treatment.CV-8688 inhibited viability of both cell types in a dose-dependentmanner. Staurosporine, the positive control, exerted 100% inhibition ofcell viability in both cell lines after 24-hour treatment. (FIGS.13A-13K).

Example 10 Cytotoxicity of Malassezin and Malassezin Derivatives—LactateDehydrogenase Release Assays

Study Summary

The LDH assay quantitatively measures lactate dehydrogenase (“LDH”)released into the media from damaged cells as a biomarker forcytotoxicity and cytolysis.

Assay Procedures

CytoTox-ONE™ Homogenous Membrane Integrity Assay was purchased fromPromega. Cell seeding, preparation of the compound source plate, andexposure of cells to test compounds were performed as described inExample 8.

For the LDH release assay, plates were removed from the incubator andequilibrated at room temperature for 15 minutes. Plates were thencentrifuged at 1,000 RPM for 1 minute. 20 μL of cell culture medium wastransferred into a new 384-well black solid plate. Then, 20 μL ofCytoTOX-ONE™was added into each well and incubated at room temperaturefor 10 minutes. Afterward, 10 μL of stop solution were added to eachwell, and the plates were agitated at 500 rpm for 1 minute. Plates wereread using an excitation wavelength of 560 nm and an emission wavelengthof 590 nm on EnSpire. The relative activity was calculated according tothe following formula:Relative Activity(%)=100%×(Lum_(sample)−Lum_(bkg))/(Lum_(vehicle)−Lum_(bkg)).Results

CV-8684 did not induce significant release in either cell line after72-hour incubation. CV-8685 showed a dose-dependent induction effect onLDH release from WM115, but not MeWo, cells after 24-hour treatment.CV-8688 induced LDH release at the highest concentration tested. (FIGS.14A-14L).

Example 11 Arylhydrocarbon Receptor Activation Potential of Malassezinand Malassezin Derivatives

Assay Procedures

HepG2-AhR-Luc cells were purchased from Pharmaron, One-Glo Luciferaseassay system was purchased from Promega, DMEM was purchased fromHyclone, and penicillin/streptomycin was purchased from Solabio.

Culture media for stably transfected HepG2 cells was prepared bysupplementing DMEM with high glucose and L-glutamine, as well as 10%FBS.

HepG2-AhR-Luc cells were cultured in T-75 flasks at 37° C., 5% CO₂, and95% relative humidity. Cells were allowed to reach 80-90% confluencebefore detachment and splitting.

Cultivated cells were rinsed with 5 mL PBS. PBS was aspirated away, 1.5mL trypsin was added to the flask, and cells were incubated at 37° C.for approximately 5 minutes or until the cells detached and floated.Trypsin was inactivated by adding excess serum-containing media.

The cell suspension was transferred to a conical tube and centrifuged at120 g for 10 minutes to pellet the cells. Cells were resuspended inseeding media at a proper density. 40 μL of cells were transferred to a384-well culture plate (5×10³ cells/well). Plates were placed in theincubator at 37° C. for 24 hours.

Afterward, stock solutions of test compounds and omeprazole positivecontrol were prepared. 40 nL of compound solutions were transferred intothe assay plate using Echo550. The plate was then placed back into theincubator for compound treatment.

Later, after 24 hours of treatment, the plate was removed from theincubator and allowed to cool at ambient temperature. 30 μL One-Gloreagent equal to that of the culture medium was added in each well.Cells were allowed to lyse for at least 3 minutes, and then measured ina luminometer.

Dose responses were graphed using the non-linear regression analysis inXLfit, and EC₅₀ values were also calculated.

Results

AhR-Luciferase assay results are shown in FIGS. 15A-15F.

Example 12 MelanoDerm™ Assays

Study Summary

The purpose of this study is to evaluate the potential dermal irritationof the test article to the MelanoDerm™ Skin Model after repeatedexposures for dose selection for a subsequent study. Toxicity will bedetermined by measuring the relative conversion of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in thetest article-treated tissues compared to the negative/solventcontrol-treated tissues.

The MelanoDerm™ Skin Model provided by MatTek Corporation (Ashland,Mass.) will be used in this study. The MelanoDerm™ tissue consists ofnormal, human-derived epidermal keratinocytes (NHEKs) and melanocytes(NHMs) which have been cultured to form a multilayered, highlydifferentiated model of the human epidermis. The NHMs within co-culturesundergo spontaneous melanogenesis leading to tissues of varying levelsof pigmentation. The cultures are grown on cell culture inserts at theair-liquid interface, allowing for topical application of skinmodulators. The MelanoDerm™ model exhibits in vivo-like morphologicaland ultrastructural characteristics. NHMs localized in the basal celllayer of MelanoDerm™ tissues are dendritic and spontaneously producemelanin granules which progressively populate the layers of the tissue.Thus the test system may be used to screen for materials which mayinhibit or stimulate the production of melanin relative to the negativecontrols.

The experimental design of this study consists of the determination ofthe pH of the neat test article if possible (and/or dosing solution asappropriate) and a definitive assay to determine the relative tissueviability after repeated exposures. The MelanoDerm™ Skin Model will beexposed to the test article for a total of 7 days. The test article willbe topically applied to the MelanoDerm™ Skin Model every 48 hours(within a timeframe of 48±2 hours from previous treatment). The toxicityof the test article will be determined by the NAD(P)H-dependentmicrosomal enzyme reduction of MTT (and, to a lesser extent, by thesuccinate dehydrogenase reduction of MTT) in control and testarticle-treated tissues. (Berridge et al., 1996). Data will be presentedin the form of relative survival (MTT conversion relative to thenegative control).

Materials

MelanoDerm™ Maintenance Medium (EPI-100-LLMM) and MelanoDerm™ Skin Model(MEL-300-A) were supplied by MatTek Corporation. 1% Kojic acid (preparedin sterile, deionized water) and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) weresupplied by Sigma. Dulbecco's Modified Eagle's Medium (DMEM) containing2 mM L-glutamine (MTT Addition Medium) was supplied by QualityBiological. Isopropanol was supplied by Aldrich. Sterile Ca⁺⁺ and Mg⁺⁺Free Dulbecco's Phosphate Buffered Saline (CMF-DPBS) was supplied byInvitrogen or equivalent. Sterile Deionized Water was supplied byQuality Biological or equivalent. DMSO was supplied by CiVenti Chem.

Assay Procedures

Test articles will generally be tested neat or as directed by theSponsor (see Protocol Attachment 1). Ten microliters (10 μL) or 25 μL ofeach test article will be applied directly on the tissue so as to coverthe upper surface. Depending on the nature of the test article (liquids,gels, creams, foams, etc.), the use of a dosing device, mesh or otheraid to allow the uniform spreading of the test article over the surfaceof the tissue may be necessary.

In the days of dosing, each test article will be diluted at least200-fold using the appropriate volume of EPI-100-LLMM (or alternatesolvent as determined during the solubility testing). A fresh dilutionin EPI-100-LLMM will be prepared for each dosing. The final dilution tobe performed for dosing solution preparation will be determined from thesolubility assessment above and documented in the study workbook.

DMSO diluted as 0.5% (v/v) in EPI-100-LLMM will be used as vehiclecontrol and dosed onto the tissues (10 μL and 25 μL doses) based on thesame procedure used for the test articles and assay controls.

The test articles will be applied topically to the MelanoDerm™ tissueevery 48 hours (within a timeframe of 48±2 hours from previoustreatment) during a 7-day trial. Ten and 25 microliters, respectively,of each test article will be applied to each tissue. Twenty fivemicroliters of the positive and negative controls, respectively, will beapplied to each tissue.

The pH of the neat liquid test article (and/or dosing solution asappropriate) will be determined, if possible. The pH will be determinedusing pH paper (for example, with a pH range of 0-14 to estimate, and/ora pH range of 5-10 to determine a more precise value). The typical pHincrements on the narrower range pH paper are approximately 0.3 to 0.5pH units. The maximum increment on the pH paper is 1.0 pH units.

The definitive assay will include a negative control and a positivecontrol. The MelanoDerm™ tissues designated to the assay negativecontrol will be treated with 25 μL of sterile, deionized water. Twentyfive microliters of 1% Kojic acid (prepared in sterile, deionized waterand filtered at the time of preparation) will be used to dose thetissues designated to the assay positive control. The 1% Kojic acid willbe stored in a tube covered with aluminum foil until used within 2 hoursof preparation. The negative and positive control exposure times will beidentical to those used for the test articles.

It is necessary to assess the ability of each test article to directlyreduce MTT. A 1.0 mg/mL MTT solution will be prepared in MTT AdditionMedium as described below. Approximately 25 μL of the test article willbe added to 1 mL of the MTT solution and the mixture incubated in thedark at 37° C.±1° C. for one to three hours. A negative control, 25 μLof sterile, deionized water, will be tested concurrently. If the MTTsolution color turns blue/purple, the test article is presumed to havereduced the MTT. Water insoluble test materials may show directreduction (darkening) only at the interface between the test article andthe medium.

The MTT direct reduction test for the test article(s) may have beenpreviously performed in an independent study. In such cases, the resultsof the MTT direct reduction test may be used for this specific study andthe initial study will be referenced.

Tissue Exposure: At least 16 hours after initiating the cultures, twoMelanoDerm™ tissues (considered untreated at Day 0) will be photographedusing a digital camera to aid in the visual assessment of the degree ofpigmentation of the tissues at time zero of the assay. The exactprocedures used to collect images of the tissues will be specified inthe study workbook and report. The MelanoDerm™ tissues will be rinsedwith CMF-DPBS, will be blotted dry on sterile absorbent paper andcleared of excess liquid. The MelanoDerm™ tissues will be transferred tothe appropriate MTT containing wells after rinsing and processed in theMTT assay as described in the MTT Assay section.

At least 16 hours after initiating the cultures, the tissues will bemoved on a new 6-well plate containing 0.9 mL of fresh, pre-warmedEPI-100-LLMM. The trial will be conducted over a 7-day timeframe. Twotissues will be treated topically on the first day, and every 48 hours(within a timeframe of 48+/−2 hours from previous treatment) with 10 and25 microliters, respectively, of each test article. The medium will berefreshed daily (within a timeframe of 24+/−2 hours from previousrefeeding); the tissues will be moved to a new 6-well plate containing0.9 mL of fresh, pre-warmed EPI-100-LLMM.

Two tissues will be treated topically on the first day, and every 48hours (within a timeframe of 48+/−2 hours from previous treatment) with25 μL of positive and negative controls, respectively. The medium willbe refreshed daily (within a timeframe of 24+/−2 hours from previousrefeeding); the tissues will be moved to a new 6-well plate containing0.9 mL of fresh, pre-warmed EPI-100-LLMM. The tissues will be incubatedat 37±1° C. in a humidified atmosphere of 5±1% CO2 in air (standardculture conditions) for the appropriate exposure times.

On the days of dosing, the MelanoDerm™ tissue will be first gentlyrinsed three times using ˜500 μL of CMF-DPBS to remove any residual testarticle. The tissues will then be moved to a new 6-well plate containing0.9 mL of fresh, pre-warmed EPI-100-LLMM and dosed with the appropriatetest article, negative or positive control. The tissues will beincubated at 37±1° C. in a humidified atmosphere of 5±1% CO2 in air(standard culture conditions) for the appropriate exposure times. Theexact rinsing procedure will be documented in the study workbook.

At the end of the 7-day trial, the MelanoDerm™ tissues dosed with thenegative or positive control, and with each test article will bephotographed using a digital camera to aid in the visual assessment ofthe degree of pigmentation of the tissues at the end of the assay (Day7). The exact procedures used to collect images of the tissues will bespecified in the study workbook and report. Then, the viability of thetissues will be determined by MTT reduction as indicated below.

MTT Assay: A 10× stock of MTT prepared in PBS (filtered at time of batchpreparation) will be thawed and diluted in warm MTT Addition Medium toproduce the 1.0 mg/mL solution no more than two hours before use. Threehundred μL of the MTT solution will be added to each designated well ofa pre-labelled 24-well plate.

After the exposure time, each MelanoDerm™ tissue designated for the MTTassay will be rinsed with CMF-DPBS, blotted dry on sterile absorbentpaper, and cleared of excess liquid. The MelanoDerm™ tissues will betransferred to the appropriate MTT containing wells after rinsing. The24-well plates will be incubated at standard conditions for 3±0.1 hours.

After 3±0.1 hours, the MelanoDerm™ tissues will be blotted on sterileabsorbent paper, cleared of excess liquid, and transferred to apre-labelled 24-well plate containing 2.0 mL of isopropanol in eachdesignated well. The plates will be covered with parafilm and stored inthe refrigerator (2-8° C.) until the last exposure time is harvested. Ifnecessary, plates may be stored overnight (or up to 24 hours after thelast exposure time is harvested) in the refrigerator prior to extractingthe MTT. Then the plates will be shaken for at least 2 hours at roomtemperature. At the end of the extraction period, the liquid within thecell culture inserts will be decanted into the well from which the cellculture insert was taken. The extract solution will be mixed and 200 μLtransferred to the appropriate wells of 96-well plate. Two hundred μL ofisopropanol will be added to the wells designated as blanks. Theabsorbance at 550 nm (OD550) of each well will be measured with aMolecular Devices Vmax plate reader (with AUTOMIX function on).

In cases where the test article is shown to reduce MTT, only testarticles that remain bound to the tissue after rinsing, resulting in afalse MTT reduction signal, present a problem. To demonstrate thatpossible residual test article is not acting to directly reduce the MTT,a functional check is performed in the definitive assay to show that thetest material is not binding to the tissue and leading to a false MTTreduction signal.

To determine whether residual test article is acting to directly reducethe MTT, a freeze-killed control tissue is used. Freeze killed tissue isprepared at IIVS by placing untreated MelanoDerm™/EpiDerm™ (Melanoderm™without melanocytes) tissues in the −20° C. freezer at least overnight,thawing to room temperature, and then refreezing. Once killed, thetissue may be stored indefinitely in the freezer. Freeze killed tissuesmay be received already prepared from MatTek Corporation, and stored inthe −20° C. freezer until use. To test for residual test articlereduction, killed tissues are treated with the test article in thenormal fashion. All assay procedures will be performed in the samemanner as for the viable tissue. At least one killed control treatedwith sterile deionized water (negative killed control) will be tested inparallel since a small amount of MTT reduction is expected from theresidual NADH and associated enzymes within the killed tissue.

If little or no MTT reduction is observed in the test article-treatedkilled control, the MTT reduction observed in the test article-treatedviable tissue may be ascribed to the viable cells. If there isappreciable MTT reduction in the treated killed control (relative to theamount in the treated viable tissue), additional steps must be taken toaccount for the chemical reduction or the test article may be considereduntestable in this system. The OD550 values from the killed controlswill be analyzed as described below

The raw absorbance data will be captured and saved as a print-file andimported into an Excel spreadsheet. The mean OD550 value of the blankwells will be calculated. The corrected mean OD550 value of the negativecontrol(s) will be determined by subtracting the mean OD550 value of theblank wells from their mean OD550 values. The corrected OD550 values ofthe individual test article exposures and the positive control exposureswill be determined by subtracting from each the mean OD550 value for theblank wells. All calculations will be performed using an Excelspreadsheet. Although the algorithms discussed are performed tocalculate the final endpoint analysis at the treatment group level, thesame calculations can be applied to the individual replicates.Corr. test article exposure OD550=Test article exposure OD550−Blank meanOD550

If killed controls (KC) are used, the following additional calculationswill be performed to correct for the amount of MTT reduced directly bytest article residues. The raw OD550 value for the negative controlkilled control will be subtracted from the raw OD550 values for each ofthe test article-treated killed controls, to determine the net OD550values of the test article-treated killed controls.Net OD550 for each test article KC=Raw OD550 test article KC−Raw OD550negative control KC

The net OD550 values represent the amount of reduced MTT due to directreduction by test article residues at specific exposure times. Ingeneral, if the net OD550 value is greater than 0.150, the net amount ofMTT reduction will be subtracted from the corrected OD550 values of theviable treated tissues to obtain a final corrected OD550 value. Thesefinal corrected OD550 values will then be used to determine the % ofControl viabilities.Final Corrected OD550=Corrected test article OD550(viable)−Net OD550test article (KC)

Finally, the following % of Control calculations will be made:% viability=[(Final corrected OD550 of Test Article or PositiveControl)/(Corrected mean OD550 of Negative Control)]×100Results

MelanoDerm™ assay results are shown in FIGS. 16A-16K. Malassezin-,compound I-, and compound II-treated tissues demonstrated reducedpigmentation on day 7 of the experiment. FIGS. 17A-17K show 15×magnification images of MelanoDerm™ samples exposed to the listedtreatment.

Example 13 Zebrafish Assays

Assay Procedures

Compounds: Compounds will be provided by Study Sponsor as Master Stock(MS) solution at the highest soluble concentration in water/PBS or DMSO.

Standard procedures for embryo collection: Phylonix AB zebrafish will begenerated by natural mating or using a Mass Embryo Production System(MEPS, Aquatic Habitats). Approximately 50 zebrafish will be generatedper female zebrafish. Zebrafish will be maintained at 28° C. in fishwater. Zebrafish will be cleaned (dead zebrafish removed) and sorted bydevelopmental stage. Because zebrafish receive nourishment from anattached yolk sac, no feeding is required for 6 days post fertilization(dpf).

Compound Solubility: Master Stock (MS) (using the highest concentration)will be diluted in pure DMSO to sub-stock solutions (SS) ie: 10, 50,100, 200, 300 mM, etc. Fish water [200 mg Instant Ocean Sea Salt(Aquarium Systems) per liter of deionized water; pH 6.6-7.0 maintainedwith 2.5 mg/liter Neutral Regulator (Seachem Laboratories Inc.);conductivity 850-950 ρS], supplied by Phylonix, will be dispensed into atesting vessel, 4 ml/vessel.

To generate test compound solution (TS), 4 μl of each SS will be addeddirectly to fish water. Example: 4 μl of 10 mM SS added to fish waterwill generate 10 μM TS; final DMSO concentration will be 0.1%.Alternatively, to obtain the same final TS and DMSO concentrations, 10μl SS can be added to 10 ml/vessel of fish water. For assays that cantolerate DMSO up to 1%, 40 μl of SS can be used to generate 100 μM TS.If 10 ml fish water is used, volume of SS should be increasedproportionally to obtain the same final TS and DMSO concentrations. Thesolution will be incubated at 28° C. for the length of time specifiedfor each assay and visually examined daily for presence ofprecipitation.

Maximum Tolerable Concentration (MTC): MTC (LCio) will be used as thestandard criterion for compound lethality, determined using 10 compoundconcentrations. After determining the highest soluble compoundconcentration, Study Sponsor will select 10 concentrations.

Thirty ˜2 dpf chorionated Phylonix wild-type AB zebrafish will bedistributed into wells of 6-well microplates containing 4 ml/well fishwater and DMSO at a concentration ranging from 0.1-1% depending oncompound solubility.

10 concentrations (i.e.: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and500 μM (or up to the concentration permitted by compound solubility),will be tested initially. If necessary, additional higher (up to 2000μM) or lower (down to 0.001 μM) concentrations will be tested.

Zebrafish will be incubated with each concentration of test compound inthe dark at 28° C. for 3 days. Untreated and 0.1-1% DMSO treatedzebrafish will be used as assay and vehicle controls. To calculate %lethality, after treatment, number of dead zebrafish will be counteddaily and removed. At 5 dpf, dead animals will be counted to calculate %lethality (=total number of dead zebrafish/30). Note, if dead zebrafishdisintegrate, number of dead zebrafish will be deduced by countingnumber of live zebrafish.

To estimate MTC, lethality curves will be generated by plotting %lethality vs concentration using EXCEL software. To obtain mean and SDof MTC, experiments will be performed 3 times.

Visually assess compound effect on zebrafish skin pigmentation:Zebrafish skin pigment cells including xanthophores, iridophores, andmelanophores (melanocytes) originate from neural crest cells. Inzebrafish, differentiated skin pigment precursor cells express pigmentat ˜24 hpf. The focus of this study is melanocytes which expressmelanin, the black pigment on the surface of the skin. Melanocytesinitially appear as small patches of black color in the dorsal headregion. As zebrafish develop, the number of patches increase and fuse toform bands which extend to the tail region. In contrast, mutant albinozebrafish exhibit sparse skin pigmentation. Compounds will beadministered at 2 dpf, to assess if compounds arrest the continuousprocess of embryonic pigmentation, which is completed by 5 dpf. Threeconcentrations, MTC, 50% MTC, and 25% MTC, will be tested for eachcompound.

Thirty 2 dpf self-hatched Phylonix wild-type AB zebrafish will betreated with each compound concentration for 3 days. Untreated and 0.1%DMSO treated zebrafish will be used as controls. Positive control:phenylthiourea (PTU, 0.03%).

Zebrafish will be visually examined daily using a dissecting lightmicroscope; compound and PTU treated zebrafish will be compared tountreated and vehicle treated control zebrafish. Number of zebrafishexhibiting decreased pigmentation will be counted daily and expressed as% of test animals; a representative image will be provided. To identifyoptimum compound concentration and treatment time for decreasedpigmentation, a kinetic curve will be generated by plotting % zebrafishexhibiting decreased skin pigmentation vs. time (dpf). Fisher's exacttest will be used to determine if compound effect is significant(P<0.05).

Additional visual assessment of compound effect on zebrafish skinpigmentation will be performed after treatment with: 0.1, 1, and 3 μM.Thirty 2 dpf self-hatched Phylonix wild-type AB zebrafish will betreated with each compound concentration for 3 days. Untreated and 0.1%DMSO treated zebrafish will be used as controls. Positive control:phenylthiourea (PTU, 0.003%). Zebrafish will be visually examined dailyusing a dissecting light microscope; compound and PTU treated zebrafishwill be compared to untreated and vehicle treated control zebrafish.

At 5 dpf, number of zebrafish exhibiting decreased pigmentation will becounted and expressed as % of test animals; a representative image willbe provided. To identify optimum compound concentration and treatmenttime for decreased pigmentation, a kinetic curve will be generated byplotting % zebrafish exhibiting decreased skin pigmentation vsconcentration. Fisher's exact test will be used to determine if compoundeffect is significant (P<0.05).

Quantitate compound effect on zebrafish skin pigmentation: Based onresults from the visual assessment, we will use the optimum conditions(concentration, compound treatment time) to quantitate compound effecton zebrafish skin pigmentation.

Twenty Phylonix wild-type AB zebrafish at the optimum stage determinedby results from the visual assessment will be treated with optimumcompound concentration. Untreated and 0.1% DMSO treated zebrafish willbe used as controls. Positive control: phenylthiourea (PTU, 0.03%).

Dorsal view image of whole zebrafish will be captured using a SPOTcamera at 2×. Dorsal head and trunk region will be defined as region ofinterest (ROI) using Adobe Photoshop selection function. Black skinpigmentation in the ROI will be highlighted using Photoshop highlightingfunction. Total pigment signal (PS) in pixels will be determined usingthe Photoshop histogram function.

If compound affects zebrafish growth, body length (L) and trunk width(W) will be smaller, which will affect ROI area and final PS. Therefore,we will normalize measurement of final signal (FS) using FS=PS/L×W.

Untreated and vehicle treated zebrafish are expected to exhibit similarFS to demonstrate that vehicle does not have an effect. PTU treatedzebrafish are expected to exhibit low FS to validate the assay. Compoundtreated zebrafish will be compared with vehicle treated controlzebrafish.

To determine if compound effect is significant (P<0.05), mean FS forcompound treated zebrafish will be compared to mean FS of vehicletreated zebrafish using Student's t test.

Additional quantitation of compound effect on zebrafish skinpigmentation will be performed after treatment with: 0.5 and 1.5 μMcompound concentration.

Twenty 2 dpf Phylonix wild-type AB zebrafish will be treated with 0.5and 1.5 μM compound concentration. Untreated and 0.1% DMSO treatedzebrafish will be used as controls. Positive control: phenylthiourea(PTU, 0.003%).

Dorsal view image of whole zebrafish will be captured using a SPOTcamera at 2×. Dorsal head region will be defined as region of interest(ROI) using Adobe Photoshop selection function. Black skin pigmentationin the ROI will be highlighted using Photoshop highlighting function.Total pigment signal (PS) in pixels will be determined using thePhotoshop histogram function.

If compound affects zebrafish growth, body length (L) will be shorterand trunk width (W) will be smaller, which will affect ROI area andfinal PS. Therefore, we will normalize final signal (FS) measurementusing FS=PS/LxW.

Untreated and vehicle treated zebrafish are expected to exhibit similarFS to confirm no effect of vehicle. PTU treated zebrafish are expectedto exhibit low FS, validating the assay. Compound treated zebrafish willbe compared with vehicle treated control zebrafish.

To determine if compound effect is significant (P<0.05), mean FS forcompound treated zebrafish will be compared to mean FS of vehicletreated zebrafish using Student's t test.

Results

Visual assessment results for zebrafish exposed to compound II are shownin FIGS. 18A-18F and FIGS. 19A-19F. A chart summarizing results from thevisual assessment portion of the study is shown in FIG. 20.

Quantitative assessment regions of interest and results for zebrafishexposed to compound II are shown in FIGS. 21A-21E and FIGS. 22A-22B.

Example 14 Stability of Malassezin and Malassezin Derivatives in DMSOand Cell Culture Media

Tested compounds were prepared at 100 μM in DMSO and culture medium. Thesolutions were incubated at room temperature for 2 hours and analyzedusing LC-MS. The peak area was used to evaluate the compound remainingin the solvent.

Results

The LC-MS results are shown in FIGS. 23A-23J. The results indicate thatthe compounds are stable in culture medium after 2-hour incubation.

DOCUMENTS

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All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is:
 1. A method for brightening skin in a subjectcomprising: contacting the subject with a compound having the structureof formula (II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a hydrate or cosmetically or pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the compound is selected fromthe group consisting of:


3. A method for inducing melanocyte apoptosis in a subject comprising:contacting the subject with a compound having the structure of formula(II):

wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 areindependently selected from the group consisting of hydrogen and methyl;or a hydrate or cosmetically or pharmaceutically acceptable saltthereof.
 4. The method of claim 3, wherein the compound is selected fromthe group consisting of:


5. The method of claim 1, wherein the subject is contacted with apharmaceutically acceptable salt of the compound having the structure offormula (II).
 6. The method of claim 1, wherein the subject is contactedwith a cosmetically acceptable salt of the compound having the structureof formula (II).
 7. The method of claim 3, wherein the subject iscontacted with a pharmaceutically acceptable salt of the compound havingthe structure of formula (II).
 8. The method of claim 3, wherein thesubject is contacted with a cosmetically acceptable salt of the compoundhaving the structure of formula (II).